(19)
(11)EP 3 194 575 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
29.04.2020 Bulletin 2020/18

(21)Application number: 15888720.8

(22)Date of filing:  11.11.2015
(51)International Patent Classification (IPC): 
C12N 5/0775(2010.01)
A61P 3/06(2006.01)
A61P 3/04(2006.01)
(86)International application number:
PCT/US2015/060176
(87)International publication number:
WO 2017/082896 (18.05.2017 Gazette  2017/20)

(54)

PROCESS AND COMPOSITIONS FOR ACHIEVING MAMMALIAN ENERGY BALANCE

VERFAHREN UND ZUSAMMENSETZUNGEN ZUR ERZIELUNG EINES ENERGIEGLEICHGEWICHTS BEI SÄUGETIEREN

PROCÉDÉ ET COMPOSITIONS PERMETTANT D'OBTENIR UN ÉQUILIBRE ÉNERGÉTIQUE CHEZ LE MAMMIFÈRE


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 10.11.2015 US 201514936830

(43)Date of publication of application:
26.07.2017 Bulletin 2017/30

(73)Proprietor: Sami Labs Limited
Bangalore 560 058 (IN)

(72)Inventors:
  • MAJEED, Muhammed
    East Windsor, NJ 08520 (US)
  • BANI, Sarang
    Bangalore 560058 (IN)
  • PANDEY, Anjali
    Bangalore 560058 (IN)
  • NAGABHUSHANAM, Kalyanam
    East Windsor, NJ 08520 (US)

(74)Representative: Dr. Gassner & Partner mbB 
Wetterkreuz 3
91058 Erlangen
91058 Erlangen (DE)


(56)References cited: : 
WO-A1-2013/188138
US-A1- 2005 158 706
US-A1- 2009 098 220
US-A1- 2015 218 935
WO-A2-00/44882
US-A1- 2005 158 706
US-A1- 2015 030 662
  
  • DATABASE MEDLINE [Online] US NATIONAL LIBRARY OF MEDICINE (NLM), BETHESDA, MD, US; January 1996 (1996-01), SCARPACE P J ET AL: "Thermogenesis in brown adipose tissue with age: post-receptor activation by forskolin.", XP002775748, Database accession no. NLM8584432 & SCARPACE P J ET AL: "Thermogenesis in brown adipose tissue with age: post-receptor activation by forskolin.", PFLUGERS ARCHIV : EUROPEAN JOURNAL OF PHYSIOLOGY JAN 1996, vol. 431, no. 3, January 1996 (1996-01), pages 388-394, ISSN: 0031-6768
  • GUO-XIAO WANG ET AL: "The brown fat secretome: metabolic functions beyond thermogenesis", TRENDS IN ENDOCRINOLOGY AND METABOLISM., vol. 26, no. 5, 1 May 2015 (2015-05-01), pages 231-237, XP055420036, US ISSN: 1043-2760, DOI: 10.1016/j.tem.2015.03.002
  • FUQIANG LI ET AL: "Protein kinase A suppresses the differentiation of 3T3-L1 preadipocytes", CELL RESEARCH - XIBAO YANJIU, vol. 18, no. 2, 1 February 2008 (2008-02-01), pages 311-323, XP055420065, GB, CN ISSN: 1001-0602, DOI: 10.1038/cr.2008.12
  • SUN ET AL.: 'Brown adipose tissue derived VEGF-A, [ modulates cold tolerance and energy expenditure.' MOL METAB vol. 3, no. 4, July 2014, pages 474 - 483, XP055381550
  • QIAN ET AL.: 'BMP4-mediated brown fat-like changes in white adipose tissue alter glucose and energy homeostasis.' PROC NAIL ACAD SCI U S A vol. 110, no. 9, 26 February 2013, pages E798 - 807, XP055381553
  • YANG ET AL.: 'cAMP/PKA Regulates Osteogenesis, Adipogenesis and Ratio of RANKUOPG mRNA Abstract - Expression in Mesenchymal Stem Cells by Suppressing Leptin.' PLOS ONE vol. 3, no. 2, 2008, page E1540, XP002676555
  • , [Online] XP055513261, Lonza Retrieved from the Internet: URL:https://www.google.de/url?sa=t&rct=j&q =&esrc=s&source=web&cd=1&ved=2ahUKEwj1yMy9 3PbdAhVHDuwKHTDUAeEQFjAAegQIBxAC&url=http% 3A%2F%2Fbio.lonza.com%2Fgo%2Fliterature%2F 5162.pdf&usg=AOvVaw1P7meX9k8FLFsovnDfcafb> [retrieved on 2018-10-08]
  • Angeliki Karamitri ET AL: "Combinatorial Transcription Factor Regulation of the Cyclic AMP-response Element on the Pgc-1 [alpha] Promoter in White 3T3-L1 and Brown HIB-1B Preadipocytes", Journal of Biological Chemistry, vol. 284, no. 31, 31 July 2009 (2009-07-31), pages 20738-20752, XP055513219, US ISSN: 0021-9258, DOI: 10.1074/jbc.M109.021766
  • Georgios Karamanlidis ET AL: "C/EBP[beta] Reprograms White 3T3-L1 Preadipocytes to a Brown Adipocyte Pattern of Gene Expression", Journal of Biological Chemistry, vol. 282, no. 34, 24 August 2007 (2007-08-24), pages 24660-24669, XP055513238, US ISSN: 0021-9258, DOI: 10.1074/jbc.M703101200
  • Kai Sun ET AL: "Brown adipose tissue derived VEGF-A modulates cold tolerance and energy expenditure", Molecular Metabolism, vol. 3, no. 4, 1 July 2014 (2014-07-01), pages 474-483, XP055381550, ISSN: 2212-8778, DOI: 10.1016/j.molmet.2014.03.010
  • Shu-Wen Qian ET AL: "BMP4-mediated brown fat-like changes in white adipose tissue alter glucose and energy homeostasis", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 110, no. 9, 26 February 2013 (2013-02-26), pages E798-E807, XP055381553, ISSN: 0027-8424, DOI: 10.1073/pnas.1215236110
  • Jun Wu ET AL: "Beige Adipocytes Are a Distinct Type of Thermogenic Fat Cell in Mouse and Human", Cell, vol. 150, no. 2, 1 July 2012 (2012-07-01), pages 366-376, XP055088017, ISSN: 0092-8674, DOI: 10.1016/j.cell.2012.05.016
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS



[0001] This application is the PCT filing drawing priority from U.S. non-provisional patent application 14936830 filed on November 10, 2015.

BACKGROUND OF THE INVENTION



[0002] Field of the invention: The invention in general relates to dietary supplements. More specifically, the present invention relates to a method of achieving optimal mammalian energy balance using forskolin on a particular physiological and developmental stage of the mammalian cellular system.

[0003] Description of Prior Art: Disruption of mammalian energy balance has been implicated as the cause for worldwide epidemics of metabolic diseases that calls for modifications in life style and food habits and also therapeutic intervention. Current diet regimens, exercise, health care awareness or drug strategies however are often unable to tackle homeostasis of energy in the mammalian body where optimally, a perfect balance between energy accumulation and energy expenditure is sought (Elattar.S and Satyanarayana, "Can Brown Fat Win the Battle against White Fat?", J Cell Physiol. 2015 Mar 11, Zafrir B, "Brown adipose tissue: research milestones of a potential player in human energy balance and obesity", Horm Metab Res. 2013 Oct;45(11):774-85). An impetus to the understanding of critical biological processes controlling brown adipocyte activity and differentiation has been in vogue in view of developing brown adipose tissue (BAT) focussed therapies for energy homeostasis (Giralt M, "White, brown, beige/brite: different adipose cells for different functions? Endocrinology. 2013 Sep; 154(9):2992-3000) where undue energy abundance is effectively countered by optimal energy expenditure.

[0004] US 2005/0158706 A1 discloses methods and compositions for a differentiation of human pre-adipocytes into adipocytes. The methods comprise incubation of isolated human pre-adipocytes in a medium containing glucose, a cyclic AMP inducer such as isobutylmethylxanthine or forskolin, a glucocorticoid or glucocorticoid analogue, insulin or an insulin analogue and a PPARĪ³ agonist or RXR agonist. The document also concerns methods for determining the ability of a compound to affect the differentiation of pre-adipocytes to adipocytes.

[0005] Li, F. et al., Cell Research (2008), vol. 18, pages 311 to 323 disclose that a stimulation of protein kinase A activity suppresses adipogenesis and inhibition of protein kinase A activity markedly accelerates the adipogenic process in cells of pre-adipocyte cell line 3T3-L1. Furthermore, the document discloses that forskolin at high concentrations can significantly increase dexamethasone and insulin-induced adipogenesis and that pretreatment of cells with forskolin inhibited adipogenesis in a dose-dependent manner.

[0006] The present disclosure relates to the potential of forskolin to mediate mammalian energy balance. Accordingly, it is the principle objective of the present disclosure to disclose,
  1. A. The ability of forskolin to prevent the formation of lipids within adult adipocytes during the differentiation of pre-adipocytes to adipocytes wherein the adipogenesis (fat deposition) inhibition is remarkably enhanced when forskolin is administered (brought into contact) to pre-adipocytes rather than to mature adipocytes;
  2. B. The ability of forskolin to enhance the expression of secreted factors that selectively recruit brown adipose tissue (BAT) like bone morphogenetic protein-7 (BMP-7), bone morphogenetic protein-4 (BMP-4), vascular endothelial growth factor (VEGF-A) and mitochondrial uncoupling protein (UCP1) wherein said enhanced expression of secreted factors that selectively recruit brown adipose tissue (BAT) is remarkably more enhanced when forskolin is administered (brought into contact) to pre-adipocytes than to mature adipocytes. In other words, forskolin treated pre-adipocytes are selectively able to differentiate into BAT.


[0007] The present invention fulfils the aforesaid objectives and provides further related advantages.

SUMMARY OF THE INVENTION



[0008] The present invention is defined in the appended claims. This disclosure relates to:
  1. (A) The ability of forskolin to prevent the formation of lipids within adult adipocytes during the differentiation of pre-adipocytes to adipocytes wherein the adipogenesis (fat deposition) inhibition is remarkably more enhanced when forskolin is administered (brought into contact) to pre-adipocytes than to mature adipocytes;
  2. (B) The ability of forskolin to enhance the expression of secreted factors that selectively recruit brown adipose tissue (BAT) like bone morphogenetic protein-7 (BMP-7), bone morphogenetic protein-4 (BMP-4), vascular endothelial growth factor-A (VEGF-A) and mitochondrial uncoupling protein (UCP1) wherein said enhanced expression of secreted factors that selectively recruit brown adipose tissue (BAT) is remarkably enhanced when forskolin is administered (brought into contact) to pre-adipocytes than to mature adipocytes. In other words, forskolin treated pre-adipocytes are selectively able to differentiate into BAT.


[0009] The advantages of the present disclosure includes the demonstration of a method to achieve mammalian energy balance using forskolin on a particular physiological and developmental stage of the mammalian cellular system wherein forskolin evinces increased potential to (i) inhibit adipogenesis; and (ii) enhance the expression of secreted factors that selectively recruit brown adipose tissue (BAT) like bone morphogenetic protein-7 (BMP-7), bone morphogenetic protein-4 (BMP-4), vascular endothelial growth factor-A (VEGF-A) and mitochondrial uncoupling protein (UCP1) when brought into contact or administered to pre-adipocytes rather than to mature adipocytes.

[0010] Other features and advantages of the present disclosure will become apparent from the following more detailed description, taken in conjunction with the accompanying images, which illustrate, by way of example, the principle of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS



[0011] 

Fig. 1 shows the graphical representation of BMP-7 in cell culture supernatant of cultured 3T3-L1 adipocytes when forskolin (50 µg/ml and 100 µg/ml) are respectively added before the differentiation of pre-adipocytes to adipocytes and after the differentiation of pre-adipocytes to adipocytes.

Fig. 2 shows the graphical representation of BMP-4 in cell culture supernatant of cultured 3T3-L1 adipocytes when forskolin (50 µg/ml and 100 µg/ml) are respectively added before the differentiation of pre-adipocytes to adipocytes and after the differentiation of pre-adipocytes to adipocytes.

Fig. 3 shows the graphical representation of VEGF-A in cell culture supernatant of cultured 3T3-L1 adipocytes when forskolin (50 µg/ml and 100 µg/ml) are respectively added before the differentiation of pre-adipocytes to adipocytes and after the differentiation of pre-adipocytes to adipocytes.

Fig. 4 shows the graphical representation of UCP1 in cell culture supernatant of cultured 3T3-L1 adipocytes when forskolin (50 µg/ml and 100 µg/ml) are respectively added before the differentiation of pre-adipocytes to adipocytes and after the differentiation of pre-adipocytes to adipocytes.


DETAILED DESCRIPTION OF THE MOST PREFERRED ASPECTS


(Figs. 1, 2, 3 and 4)



[0012] In the most preferred aspect, the present disclosure relates to a method of achieving mammalian energy balance using forskolin in a process of adipogenesis inhibition wherein forskolin is added separately to pre-adipocytes before differentiation and also to mature adipocytes to comparatively evaluate adipogenesis inhibition potential of, said process comprising steps of:
  1. a) Seeding mammalian adipocyte precursor cells (pre-adipocytes) in wells of microplates wherein approximately 60×104 cells are seeded for 48-72 hours to get 70-80% confluence;
  2. b) Adding forskolin at concentrations of 50 µg/ml and 100 µg/ml in the pre-seeded microplates of step a containing undifferentiated pre-adipocytes;
  3. c) Adding 200 µl of freshly prepared Adipogenesis induction medium to the wells;
  4. d) Adding 200 µl of freshly prepared Adipogenesis progression medium after 72 hours of incubation with the Adipogenesis induction medium in step c;
  5. e) Incubating the cells treated with forskolin (step b), adipogenesis induction medium (step c) and adipogenesis progression medium (step d) for 48 hours in a humidified atmosphere (37 deg. C.) of 5% CO2 and 95% air;
  6. f) Fixing the cells of step e by adding 100 µl of 10% formalin and staining using the Oil Red O technique;
  7. g) Reading the optical density of cells of step f at 492 nm in a microplate reader and expressing the results as inhibitory concentration (IC50) values using the graph pad prism software;
  8. h) Calculating the percentage inhibition of adipogenesis in the cells of steps f and g using the formula, C-T/T×100, wherein C is the absorbance of Oil Red O in differentiating/undifferentiated cells and T is the absorbance of Oil Red O in sample treated differentiating/undifferentiated cells.
  9. i) Adding 200 µl of freshly prepared Adipogenesis induction medium to the wells of step a.
  10. j) Adding 200 µl of freshly prepared Adipogenesis progression medium comprising graded concentrations of forskolin (50 µg/ml and 100 µg/ml respectively) to the wells of step i after 72 hours of incubation with the Adipogenesis induction medium;
  11. k) Incubating the cells treated with forskolin (step j), adipogenesis induction medium (step i) and adipogenesis progression medium (step j) for 48 hours in a humidified atmosphere (37 deg. C.) of 5% CO2 and 95% air;
  12. l) Fixing the cells of step I by adding 100 µl of 10% formalin and staining using the Oil Red O technique;
  13. m) Reading the optical density of cells of step m at 492 nm in a microplate reader and expressing the results as inhibitory concentration (IC50) values using the graph pad prism software;
  14. n) Calculating the percentage inhibition of adipogenesis in the cells of steps m and n using the formula, C-T/T×100, wherein C is the absorbance of Oil Red O in differentiating/undifferentiated cells and T is the absorbance of Oil Red O in sample treated differentiating/undifferentiated cells; and
  15. o) Comparing percentage inhibition of adipogenesis in the cells of steps h and o.


[0013] In another most preferred aspect, the present disclosure also relates to a method of mammalian energy balance using forskolin in a process of promoting the expression of secreted factors that selectively recruit brown adipose tissue (BAT) like bone morphogenetic protein-7 (BMP-7), bone morphogenetic protein-4 (BMP-4), vascular endothelial growth factor-A (VEGF-A) and mitochondrial uncoupling protein (UCP1) wherein said expression of secreted factors that selectively recruit brown adipose tissue (BAT) is remarkably enhanced as measured when forskolin is administered (brought into contact) to pre-adipocytes than to mature adipocytes, said method incorporating the steps of ,
  1. a) Seeding mammalian adipocyte precursor cells (pre-adipocytes) in wells of microplates wherein approximately 60×104 cells are seeded for 48-72 hours to get 70-80% confluence;
  2. b) Adding forskolin at concentrations of 50 µg/ml and 100 µg/ml in the pre-seeded microplates of step a containing undifferentiated pre-adipocytes;
  3. c) Adding 200 µl of freshly prepared Adipogenesis induction medium to the wells;
  4. d) Adding 200 µl of freshly prepared Adipogenesis progression medium after 72 hours of incubation with the Adipogenesis induction medium in step c;
  5. e) Incubating the cells treated with forskolin (step b), adipogenesis induction medium (step c) and adipogenesis progression medium (step d) for 48 hours in a humidified atmosphere (37 deg. C.) of 5% CO2 and 95% air;
  6. f) Quantitatively determining by appropriate immunoassay techniques the expressions of BMP-7, BMP-4, VEGF-A and UCP-1 in the cell supernatant;
  7. g) Adding 200 µl of freshly prepared Adipogenesis induction medium to the wells of step a;
  8. h) Adding 200 µl of freshly prepared Adipogenesis progression medium comprising graded concentrations of forskolin (50 µg/ml and 100 µg/ml respectively) to the wells of step g after 72 hours of incubation with the Adipogenesis induction medium;
  9. i) Incubating the cells treated with forskolin (step h), adipogenesis induction medium (step g) and adipogenesis progression medium (step h) for 48 hours in a humidified atmosphere (37 deg. C.) of 5% CO2 and 95% air; and
  10. j) Quantitatively determining by appropriate immunoassay techniques the expressions of BMP-7, BMP-4, VEGF-A and UCP-1 in the cell supernatant.


[0014] The present disclosure also relates to a method of achieving energy balance in mammalian adipose cellular systems, said method comprising step of administering forskolin in effective amounts targeted towards mammalian pre-adipocytes to achieve effects of (a) increased inhibition of adipogenesis and (b) increased expression of secretory factors that function individually or in combination to specifically recruit brown adipocytes or brown like (beige or brite) adipocytes. In specific embodiments, the secretory factors are selected from the group consisting of bone morphogenetic protein-7 (BMP-7), bone morphogenetic protein-4 (BMP-4), vascular endothelial growth factor-A (VEGF-A) and mitochondrial uncoupling protein (UCP1).

[0015] The present disclosure also relates to forskolin for use in therapy for obesity wherein said therapy involves achieving energy balance in mammalian adipocytes by administering forskolin in effective amounts targeted towards mammalian pre-adipocytes to bring about the effects of (a) increased inhibition of adipogenesis and (b) increased expression of secretory factors that function individually or in combination to specifically recruit brown adipocytes or brown like (beige or brite) adipocytes. In specific embodiments, the secretory factors are selected from the group consisting of bone morphogenetic protein-7 (BMP-7), bone morphogenetic protein-4 (BMP-4), vascular endothelial growth factor-A (VEGF-A) and mitochondrial uncoupling protein (UCP1)

[0016] The present disclosure also relates to a method to induce the brown like phenotype (beige or brite adipocytes) in white adipocyte depots in mammals said method comprising step of administering effective amount of forskolin to obese mammals with depots of fully differentiated white adipocytes to achieve effect of increase in secretory factors that bring about the development of brown like phenotype (beige or brown adipocytes) within white adipocyte depots. In specific embodiment, the secretory factors are vascular endothelial growth factor-A (VEGF-A) and mitochondrial uncoupling protein (UCP1). The present disclosure also relates to Forskolin for use in the therapy of obesity characterised in that forskolin is administered in effective amounts targeting mammalian white adipocyte depots to achieve effect of increased expression of secretory factors vascular endothelial growth factor-A (VEGF-A) and mitochondrial uncoupling protein (UCP1) that cause the development of brown like phenotype (beige or brite adipocytes) in white adipocyte depots in mammals.

[0017] The present disclosure also relates to a method of achieving energy balance in mammalian adipose cellular systems, said method comprising step of administering forskolin in effective amounts targeted towards mammalian pre-adipocytes to bring about of the effect of enhanced expression of mitochondria uncoupling protein 1 (UCP-1) to result in increased mitochondrial thermiogenesis in differentiated brown adipocytes and brown like (beige or brite) adipocytes.

[0018] The present disclosure also relates to forskolin for use in therapy for obesity wherein said therapy involves achieving energy balance in mammalian adipocytes by administering forskolin in effective amounts targeted towards mammalian pre-adipocytes to bring about the effects of enhanced expression of secretory factor mitochondria uncoupling protein 1 (UCP-1) to result in increased mitochondrial thermiogenesis in differentiated brown adipocytes and brown like (beige or brite) adipocytes.

ILLUSTRATIVE EXAMPLES



[0019] As illustrative examples of the most preferred aspects outlined herein above in paragraphs [0011]-[0017], the following results are presented to show that forskolin when administered in increasing concentration is more effective in (a) preventing adipogenesis and (b) also in promoting the expression of secreted factors like BMP-7, BMP-4, VEGF-A and UCP-1 that recruit the brown adipocytes thereby creating energy balance in mammalian cell systems, when administered at the pre-adipocyte stage than once the transformation of pre-adipocytes to adipocytes has occurred.

RESULT 1-Prevention of adipogenesis



[0020] 
Table A
CONCENTRATION (µg/ml)% inhibition of adipogenesis when forskolin is added at the pre-adipocyte stage (before differentiation into the adipocyte stage)% inhibition of adipogenesis when forskolin is added after differentiation of pre-adipocytes to the adipocyte stage
6.25 10.2 1.2
12.50 12.8 6.8
25 19.7 10.6
50 35.5 12.9
100 41.8 18.5


[0021] Table A shows that at each tested concentration of forskolin, the administration of forskolin at the mammalian pre-adipocyte stage has a profound effect on preventing adipogenesis that when administered after the differentiation of pre-adipocytes to adipocytes. Double or more than double the % inhibition of adipogenesis was observed when forskolin was administered at the pre-adipocyte stage as compared to administration at the adipocyte stage.

RESULT 2-Expression of secretory proteins that recruit brown adipocytes


A. BMP-7



[0022] The biological role of BMP-7 as a recruiter of the brown adipocyte lineage has been discussed in the following scientific literature.
  1. 1. Mathew Harms and Patrick Seale, "Brown and beige fat: development, function and therapeutic potential", Nature Medicine, Volume 19, Number 10, October 2013, pages 1252-1263;
  2. 2. BMP7 Activates Brown Adipose Tissue and Reduces Diet-Induced Obesity at Sub thermoneutrality .Mariëtte R. Boon Published: September 16, 2013; PLOS One.
  3. 3. New role of bone morphogenetic protein 7 in brown adipogenesis and energy expenditure. Tseng et al. Nature. 2008 Aug 21; 454(7207): 1000-4. doi: 10.1038/nature07221.
  4. 4. Transcriptional Control of Brown Fat Development; Kajimure et al. Cell Metabolism; Volume 11, Issue 4, 7 April 2010, Pages 257-262.


[0023] Immunoassays (Enzyme linked immunosorbent assay) for the quantification of BMP-7 in the cell culture supernatant when forskolin (50 µg/ml and 100 µg/ml) was administered at the pre-adipocyte stage and once the differentiation to adipocytes occurred indicated that forskolin profoundly increased BMP-7 expression in at the pre-adipocyte stage than at the adipocyte stage.

[0024] Thus, in correlation with the literature cited above, it may be deduced that forskolin evinces greater potential for brown fat conversion of pre-adipocytes (Fig. 1) rather than fully differentiated white adipocytes. The example exemplified by Fig. 1 provides substantiation to the disclosed most preferred embodiment that forskolin directs the selective differentiation of mammalian pre-adipocytes to brown adipocytes by allowing the expression of secretory factor BMP-7.

B. BMP-4



[0025] Acting along with BMP-7, BMP-4 is a new adipokine and acts on adipogenesis and white to brown transition (Qian S Wet al Proc Natl Acad Sci USA 110: E798-807, 2013). Immunoassays (Enzyme linked immunosorbent assay) for the quantification of BMP-4 in the cell culture supernatant when forskolin (50 µg/ml and 100 µg/ml) was administered at the pre-adipocyte stage and once the differentiation to adipocytes occurred indicated that forskolin profoundly increased BMP-4 expression in at the pre-adipocyte stage than at the adipocyte stage.

[0026] Thus, in correlation with the literature cited above, it may be deduced that forskolin evinces greater potential for conversion of white pre-adipocytes to the brite/beige adipocyte (brown adipocyte like) (Fig. 2) by the combined increased expressions and biological actions of secretory factors BMP-4 and BMP-7. The example exemplified by Fig. 2 provides substantiation to the most preferred embodiment that forskolin brings about the transformation of white pre-adipocytes to brite or beige adipocytes.

C. VEGF-A



[0027] VEGF-A over expression leads to an increase in brown adipose tissue (BAT) thermogenesis and also promotes a "BAT-like" phenotype in white adipose tissue depots. In diet-induced obese mice, introducing VEGF-A locally in BAT rescues capillary rarefaction, ameliorates brown adipocyte dysfunction, and improves deleterious effects on glucose and lipid metabolism caused by a high-fat diet challenge. These results demonstrate a direct positive role of VEGF-A in the activation and expansion of BAT. VEGF-A over expression also exerts its action on macrophages by increasing the recruitment of M2 anti-inflammatory macrophages to fat depots. The decreased obesity and the anti-inflammatory milieu induced by VEGF-A in adipose tissue is responsible for the reduction of insulin resistance in transgenic mice (Bagchi et al, "Vascular endothelial growth factor is important for brown adipose tissue development and maintenance", FASEB J. 27, 3257-3271 (2013). Immunoassays (Enzyme linked immunosorbent assay) for the quantification of VEGF-A in the cell culture supernatant when forskolin (50 µg/ml and 100 µg/ml) was administered at the pre-adipocyte stage and once the differentiation to adipocytes occurred indicated that forskolin profoundly increased VEGF-A expression in the pre-adipocyte stage than at the adipocyte stage. Thus, in correlation with the literature cited above, it may be deduced that forskolin evinces greater potential for conversion of white pre-adipocytes to the brown adipocyte like (brite or beige) cells (Fig. 3) among white adipocyte depots in the mammalian body.

D. Uncoupling Protein-1(UCP-1)



[0028] A system of thermogenesis that evolved to protect the body from hypothermia is based upon the uncoupling of oxidative phosphorylation in brown adipocytes by the mitochondrial uncoupling protein (UCP-1). It has been shown that up-regulation of UCP1 by genetic manipulations or pharmacological agents can reduce obesity and improve insulin sensitivity (International Journal of Obesity (2008) 32, S32-S38; doi:10.1038/ijo.2008.236 UCP1: its involvement and utility in obesity. L P Kozak and R Anunciado-Koza). Immunoassays (Enzyme linked immunosorbent assay) for the quantification of UCP-1 in the cell culture supernatant when forskolin (50 µg/ml and 100 µg/ml) was administered at the pre-adipocyte stage and once the differentiation to adipocytes occurred indicated that forskolin profoundly increased UCP-1 expression in the pre-adipocyte stage than at the adipocyte stage. Thus, in correlation with the literature cited above, it may be deduced that forskolin evinces greater potential for conversion of pre-adipocytes to the BAT-like or brown adipocytes and enhanced UCP-1 expression in these cells can be expected to enhance bringing about energy balance through appropriate energy expenditure (Fig. 4).

[0029] It is already reported that administration of forskolin in humans apparently does not cause clinically significant side effects (Shonteh Henderson et al, Effect of Coleus forskolii supplementation on body composition and haematological profiles in mildly overweight women, J Int Soc Sports Nutr. 2005; 2(2): 54-62). The study elucidates that supplementation with forskolin dietary supplement Forslean® [250 mg of 10% Coleus forskolii extract, 25 mg of forskolin] two times a day for 12 weeks apparently had no clinical side effects. It may thus be inferred that the illustrative in-vitro examples included herein above to achieve energy balance in mammalian adipocyte systems is also applicable in vivo studies in animals (mammals) including human beings.


Claims

1. A method of comparatively evaluating adipogenesis inhibition potential of forskolin, wherein forskolin is added separately to pre-adipocytes before differentiation and also to mature adipocytes, said process comprising steps of:

a) Seeding mammalian adipocyte precursor cells (pre-adipocytes) in wells of microplates, wherein approximately 60×104 cells are seeded for 48-72 hours to get 70-80% confluence;

b) Adding forskolin at concentrations of 50 µg/ml and 100 µg/ml, respectively, in the pre-seeded microplates of step a containing undifferentiated pre-adipocytes;

c) Adding 200 µl of freshly prepared adipogenesis induction medium to the wells;

d) Adding 200 µl of freshly prepared adipogenesis progression medium after 72 hours of incubation with the adipogenesis induction medium in step c;

e) Incubating the cells treated with forskolin (step b), adipogenesis induction medium (step c) and adipogenesis progression medium (step d) for 48 hours in a humidified atmosphere of 5% CO2 and 95% air at 37 deg. C;

f) Fixing the cells of step e by adding 100 µl of 10% formalin and staining using the Oil Red O technique;

g) Reading the optical density of cells of step f at 492 nm in a microplate reader and expressing the results as inhibitory concentration (IC50) values using the graph pad prism software;

h) Calculating the percentage inhibition of adipogenesis in the cells of steps f and g using the formula, C-T/T×100, wherein C is the absorbance of Oil Red O in differentiating/undifferentiated cells and T is the absorbance of Oil Red O in sample treated differentiating/undifferentiated cells;

i) Adding 200 µl of freshly prepared adipogenesis induction medium to the wells of step a, which wells comprise the cells;

j) Adding 200 µl of freshly prepared adipogenesis progression medium comprising graded concentrations of forskolin (50 µg/ml and 100 µg/ml respectively) to the wells of step i after 72 hours of incubation with the adipogenesis induction medium;

k) Incubating the cells treated with forskolin (step j), adipogenesis induction medium (step i) and adipogenesis progression medium (step j) for 48 hours in a humidified atmosphere of 5% CO2 and 95% air at 37 deg. C;

l) Fixing the cells of step k by adding 100 µl of 10% formalin and staining using the Oil Red O technique;

m) Reading the optical density of cells of step I at 492 nm in a microplate reader and expressing the results as inhibitory concentration (IC50) values using the graph pad prism software;

n) Calculating the percentage inhibition of adipogenesis in the cells of steps I and m using the formula, C-T/T×100, wherein C is the absorbance of Oil Red O in differentiating/undifferentiated cells and T is the absorbance of Oil Red O in sample treated differentiating/undifferentiated cells; and

o) Comparing percentage inhibition of adipogenesis in the cells of steps h and n.


 
2. A method of determining the expression of secreted factors that selectively recruit brown adipose tissue (BAT), wherein forskolin is added separately to pre-adipocytes before differentiation and also to mature adipocytes, said method incorporating the steps of:

a) Seeding mammalian adipocyte precursor cells (pre-adipocytes) in wells of microplates, wherein approximately 60×104 cells are seeded for 48-72 hours to get 70-80% confluence;

b) Adding forskolin at concentrations of 50 µg/ml and 100 µg/ml, respectively, in the pre-seeded microplates of step a containing undifferentiated pre-adipocytes;

c) Adding 200 µl of freshly prepared adipogenesis induction medium to the wells;

d) Adding 200 µl of freshly prepared adipogenesis progression medium after 72 hours of incubation with the adipogenesis induction medium in step c;

e) Incubating the cells treated with forskolin (step b), adipogenesis induction medium (step c) and adipogenesis progression medium (step d) for 48 hours in a humidified atmosphere of 5% CO2 and 95% air at 37 deg. C;

f) Quantitatively determining by appropriate immunoassay techniques the expressions of bone morphogenetic protein-7 (BMP-7), bone morphogenetic protein-4 (BMP-4), vascular endothelial growth factor-A (VEGF-A) and mitochondrial uncoupling protein (UCP-1) in the cell supernatant;

g) Adding 200 µl of freshly prepared adipogenesis induction medium to the wells of step a, which wells comprise the cells;

h) Adding 200 µl of freshly prepared adipogenesis progression medium comprising graded concentrations of forskolin (50 µg/ml and 100 µg/ml respectively) to the wells of step g after 72 hours of incubation with the adipogenesis induction medium;

i) Incubating the cells treated with forskolin (step h), adipogenesis induction medium (step g) and adipogenesis progression medium (step h) for 48 hours in a humidified atmosphere of 5% CO2 and 95% air at 37 deg. C; and

j) Quantitatively determining by appropriate immunoassay techniques the expressions of BMP-7, BMP-4, VEGF-A and UCP-1 in the cell supernatant.


 


Ansprüche

1. Verfahren zum komparativen Evaluieren eines Adipogenese-Inhibitionspotentials von Forskolin, wobei Forskolin separat zu Präadipozyten vor Differenzierung und auch zu reifen Adipozyten zugesetzt wird, wobei das Verfahren folgende Schritte umfasst:

a) Aussäen von Säugeradipozyten-Vorläuferzellen (Präadipozyten) in Vertiefungen von Mikrotiterplatten, wobei ungefähr 60×104 Zellen für 48-72 Stunden ausgesät werden, um 70-80% Konfluenz zu erreichen,

b) Zusetzen von Forskolin in Konzentrationen von jeweils 50 µg/ml und 100 µg/ml in die Vorbesäten Mikrotiterplatten von Schritt a, enthaltend undifferenzierte Präadipozyten,

c) Zusetzen von 200 µl frisch hergestelltem Adipogenese-Induktionsmedium zu den Vertiefungen,

d) Zusetzen von 200 µl frisch hergestelltem Adipogenese-Progressionsmedium nach 72 Stunden der Inkubation mit dem Adipogenese-Induktionsmedium in Schritt c,

e) Inkubation der mit Forskolin (Schritt b), Adipogenese-Induktionsmedium (Schritt c) und Adipogenese-Progressionsmedium (Schritt d) behandelten Zellen für 48 Stunden in einer befeuchteten Atmosphäre von 5% CO2 und 95% Luft bei 37°C,

f) Fixieren der Zellen von Schritt e durch Zusetzen von 100 µl von 10% Formalin und Färben unter Verwendung der Ölrot O-Technik,

g) Lesen der optischen Dichte von Zellen von Schritt f bei 492 nm in einem Mikroplattenleser und Wiedergeben der Ergebnisse als Hemmungskonzentration (IC50)-Werte unter Verwendung der Graph Pad Prism-Software,

h) Berechnen der prozentualen Hemmung von Adipogenese in den Zellen der Schritte f und g unter Verwendung der Formel C-T/T×100, wobei C die Absorption von Ölrot O in differenzierenden/undifferenzierten Zellen ist und T die Absorption von Ölrot O in Probe-behandelten differenzierenden/undifferenzierten Zellen ist,

i) Zusetzen von 200 µl frisch hergestelltem Adipogenese-Induktionsmedium zu den Vertiefungen von Schritt a, welche Vertiefungen die Zellen umfassen,

j) Zusetzen von 200 µl frisch hergestelltem Adipogenese-Progressionsmedium, umfassend abgestufte Konzentrationen von Forskolin (jeweils 50 µg/ml und 100 µg/ml) zu den Vertiefungen von Schritt i nach 72 Stunden der Inkubation mit dem Adipogenese-Induktionsmedium,

k) Inkubieren der mit Forskolin (Schritt j), Adipogenese-Induktionsmedium (Schritt i) und Adipogenese-Progressionsmedium (Schritt j) behandelten Zellen für 48 Stunden in einer befeuchteten Atmosphäre von 5% CO2 und 95% Luft bei 37°C,

l) Fixieren der Zellen von Schritt k durch Zusetzen von 100 µl von 10% Formalin und Färben unter Verwendung der Ölrot O-Technik,

m) Lesen der optischen Dichte von Zellen von Schritt I bei 492 nm in einem Mikroplattenleser und Wiedergeben der Ergebnisse als Hemmungskonzentration (IC50)-Werte unter Verwendung der Graph Pad Prism-Software,

n) Berechnen der prozentualen Hemmung von Adipogenese in den Zellen der Schritte I und m unter Verwendung der Formel C-T/Tx100, wobei C die Absorption von Ölrot O in differenzierenden/undifferenzierten Zellen ist und T die Absorption von Ölrot O in Probe-behandelten differenzierenden/undifferenzierten Zellen ist und

o) Vergleichen der prozentualen Hemmung von Adipogenese in den Zellen der Schritte h und n.


 
2. Verfahren zum Bestimmen der Expression sekretierter Faktoren, welche selektiv braunes Fettgewebe (BAT) rekrutieren, wobei Forskolin separat zu Präadipozyten vor Differenzierung und auch zu reifen Adipozyten zugesetzt wird, wobei das Verfahren die folgenden Schritte beinhaltet:

a) Aussäen von Säugeradipozyten-Vorläuferzellen (Präadipozyten) in Vertiefungen von Mikrotiterplatten, wobei ungefähr 60×104 Zellen für 48-72 Stunden ausgesät werden, um 70-80% Konfluenz zu erreichen,

b) Zusetzen von Forskolin in Konzentrationen von jeweils 50 µg/ml und 100 µg/ml in die Vorbesäten Mikrotiterplatten von Schritt a, enthaltend undifferenzierte Präadipozyten,

c) Zusetzen von 200 µl frisch hergestelltem Adipogenese-Induktionsmedium zu den Vertiefungen,

d) Zusetzen von 200 µl frisch hergestelltem Adipogenese-Progressionsmedium nach 72 Stunden der Inkubation mit dem Adipogenese-Induktionsmedium in Schritt c,

e) Inkubation der mit Forskolin (Schritt b), Adipogenese-Induktionsmedium (Schritt c) und Adipogenese-Progressionsmedium (Schritt d) behandelten Zellen für 48 Stunden in einer befeuchteten Atmosphäre von 5% CO2 und 95% Luft bei 37°C,

f) quantitatives Bestimmen der Expressionen von knochenmorphogenetischem Protein-7 (BMP-7), knochenmorphogenetischem Protein-4 (BMP-4), vaskulärem endothelialem Wachstumsfaktor-A (VEGF-A) und mitochondrialem Entkopplungsprotein (UCP-1) im Zellüberstand durch geeignete Immunassay-Techniken,

g) Zusetzen von 200 µl frisch hergestelltem Adipogenese-Induktionsmedium zu den Vertiefungen von Schritt a, welche Vertiefungen die Zellen umfassen,

h) Zusetzen von 200 µl frisch hergestelltem Adipogenese-Progressionsmedium, umfassend abgestufte Konzentrationen von Forskolin (jeweils 50 µg/ml und 100 µg/ml) zu den Vertiefungen von Schritt g) nach 72 Stunden der Inkubation mit dem Adipogenese-Induktionsmedium,

i) Inkubieren der mit Forskolin (Schritt h), Adipogenese-Induktionsmedium (Schritt g) und Adipogenese-Progressionsmedium (Schritt h) behandelten Zellen für 48 Stunden in einer befeuchteten Atmosphäre von 5% CO2 und 95% Luft bei 37°C und

j) quantitatives Bestimmen der Expressionen von BMP-7, BMP-4, VEGF-A und UCP-1 in dem Zellüberstand durch geeignete Immunassay-Techniken.


 


Revendications

1. Procédé d'évaluation comparative du potentiel d'inhibition d'adipogenèse de la forskoline, dans lequel de la forskoline est ajoutée séparément à des pré-adipocytes avant différenciation et également à des adipocytes matures, ledit processus comprenant des étapes consistant à :

a) ensemencer des cellules précurseurs d'adipocyte mammifère (pré-adipocytes) dans des puits de microplaques, dans lequel approximativement 60x104 cellules sont ensemencées pour 48 à 72 heures pour obtenir 70 à 80 % de confluence ;

b) ajouter de la forskoline à des concentrations de 50 µg/ml et 100 µg/ml, respectivement, dans les microplaques ensemencées au préalable de l'étape a contenant des pré-adipocytes non différenciés ;

c) ajouter 200 µl de milieu d'induction d'adipogenèse fraîchement préparé aux puits ;

d) ajouter 200 µl de milieu de progression d'adipogenèse fraîchement préparé après 72 heures d'incubation avec le milieu d'induction d'adipogenèse à l'étape c ;

e) incuber les cellules traitées avec la forskoline (étape b), le milieu d'induction d'adipogenèse (étape c) et le milieu de progression d'adipogenèse (étape d) pendant 48 heures dans une atmosphère humidifiée de 5 % de CO2 et 95 % d'air à 37 degrés C ;

f) fixer les cellules de l'étape e en ajoutant 100 µl de formaline à 10 % et colorer en utilisant la technique Oil Red O ;

g) lire la densité optique de cellules de l'étape f à 492 nm dans un lecteur de microplaque et exprimer les résultats en tant que valeurs de concentration d'inhibition (CI50) en utilisant le logiciel GraphPad Prism ;

h) calculer le pourcentage d'inhibition d'adipogenèse dans les cellules des étapes f et g en utilisant la formule, C-T/Tx100, dans laquelle C est l'absorbance de Oil Red O dans des cellules en différenciation/non différenciées et T est l'absorbance de Oil Red O dans des cellules en différenciation/non différenciées traitées de l'échantillon ;

i) ajouter 200 µl de milieu d'induction d'adipogenèse fraîchement préparé aux puits de l'étape a, lesquels puits comprennent les cellules ;

j) ajouter 200 µl de milieu de progression d'adipogenèse fraîchement préparé comprenant des concentrations échelonnées de forskoline (50 µg/ml et 100 µg/ml respectivement) aux puits de l'étape i après 72 heures d'incubation avec le milieu d'induction d'adipogenèse ;

k) incuber les cellules traitées avec la forskoline (étape j), le milieu d'induction d'adipogenèse (étape i) et le milieu de progression d'adipogenèse (étape j) pendant 48 heures dans une atmosphère humidifiée de 5 % de CO2 et 95 % d'air à 37 degrés C ;

l) fixer les cellules de l'étape k en ajoutant 100 µl de formaline à 10 % et colorer en utilisant la technique Oil Red O ;

m) lire la densité optique de cellules de l'étape I à 492 nm dans un lecteur de microplaque et exprimer les résultats en tant que valeurs de concentration d'inhibition (CI50) en utilisant le logiciel GraphPad Prism ;

n) calculer le pourcentage d'inhibition d'adipogenèse dans les cellules des étapes I et m en utilisant la formule, C-T/Tx100, dans laquelle C est l'absorbance de Oil Red O dans des cellules en différenciation/non différenciées et T est l'absorbance de Oil Red O dans des cellules en différenciation/non différenciées traitées de l'échantillon ; et

o) comparer le pourcentage d'inhibition d'adipogenèse dans les cellules des étapes h et n.


 
2. Procédé de détermination de l'expression de facteurs secrétés qui recrutent sélectivement du tissu adipeux brun (BAT), dans lequel de la forskoline est ajoutée séparément à des pré-adipocytes avant différenciation et également à des adipocytes matures, ledit procédé incorporant les étapes consistant à :

a) ensemencer des cellules précurseurs d'adipocyte mammifère (pré-adipocytes) dans des puits de microplaques, dans lequel approximativement 60x104 cellules sont ensemencées pour 48 à 72 heures pour obtenir 70 à 80 % de confluence ;

b) ajouter de la forskoline à des concentrations de 50 µg/ml et 100 µg/ml, respectivement, dans les microplaques ensemencées au préalable de l'étape a contenant des pré-adipocytes non différenciés ;

c) ajouter 200 µl de milieu d'induction d'adipogenèse fraîchement préparé aux puits ;

d) ajouter 200 µl de milieu de progression d'adipogenèse fraîchement préparé après 72 heures d'incubation avec le milieu d'induction d'adipogenèse à l'étape c ;

e) incuber les cellules traitées avec la forskoline (étape b), le milieu d'induction d'adipogenèse (étape c) et le milieu de progression d'adipogenèse (étape d) pendant 48 heures dans une atmosphère humidifiée de 5 % de CO2 et 95 % d'air à 37 degrés C ;

f) déterminer quantitativement par des techniques d'immunoessai appropriées les expressions de la protéine morphogénétique osseuse 7 (BMP-7), de la protéine morphogénétique osseuse 4 (BMP-4), du facteur de croissance de l'endothélium vasculaire A (VEGF-A) et de la protéine découplante mitochondriale (UCP-1) dans le surnageant cellulaire ;

g) ajouter 200 µl de milieu d'induction d'adipogenèse fraîchement préparé aux puits de l'étape a, lesquels puits comprennent les cellules ;

h) ajouter 200 µl de milieu de progression d'adipogenèse fraîchement préparé comprenant des concentrations échelonnées de forskoline (50 µg/ml et 100 µg/ml respectivement) aux puits de l'étape g après 72 heures d'incubation avec le milieu d'induction d'adipogenèse ;

i) incuber les cellules traitées avec la forskoline (étape h), le milieu d'induction d'adipogenèse (étape g) et le milieu de progression d'adipogenèse (étape h) pendant 48 heures dans une atmosphère humidifiée de 5 % de CO2 et 95 % d'air à 37 degrés C ; et

j) déterminer quantitativement par des techniques d'immunoessai appropriées les expressions de BMP-7, BMP-4, VEGF-A et UCP-1 dans le surnageant cellulaire.


 




Drawing

















Cited references

REFERENCES CITED IN THE DESCRIPTION



This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description




Non-patent literature cited in the description